Urokinase-Type Plasminogen Activator
The biochemistry of uPA has been reviewed previously (1). It is a serine protease, which is synthesized as an approximately 50 kD glycosylated single polypeptide chain pro-enzyme, pro-uPA, that is virtually catalytically inactive. The human uPA gene is located on chromosome 10 and is transcribed into a 2.5 kb long mRNA. Pro-uPA is converted into active uPA, consisting of two polypeptide chains (A and B) held together by a disulphide bond, the A-chain arising from the amino-terminal part of pro-uPA, and the B-chain arising from the carboxy-terminal part. The A-chain consists of two structural domains, a growth factor domain with homology to EGF, and a kringle domain (2). The B-chain is homologous to the catalytic part of other serine proteases, such as trypsin, chymotrypsin, and plasmin. Two-chain uPA can, by the metalloproteinase matrilysin (or PUMP-1) (3), be converted into a 33 kD catalytic active form of uPA consisting of the B-chain and the carboxy-terminal part of the A-chain (low molecular weight uPA), and a 17 kD non-catalytic fragment consisting of the N-terminal part of the A-chain.
uPA cleaves a single peptide bond in plasminogen, converting it into plasmin, that degrades a broad spectrum of proteins, including fibronectin, fibrin, and laminin (for a review see ref.(1)). In addition, plasmin activates latent forms of some metalloproteases (4) and affects various growth factor systems, e.g. by activating latent TGF-.beta. (5,6) and dissociating IGF-I from its binding protein (7) and bFGF from the surface of some cell types (8).
Many cytokines and hormones control the expression of uPA in a cell specific way (see reference (9)).
uPA is produced by many cultured cell types of neoplastic origin. It has been found that explants of tumour tissue released more uPA than the corresponding normal tissue. uPA has been identified in extracts from human lung, colon, endometrial, breast, prostate and renal carcinomas, human melanomas, murine mammary tumours, the murine Lewis lung tumour, and in ascites from human peritoneal carcinomatosis. An immunohistochemical study of invasively growing and metastasing Lewis lung carcinomas in mice consistently showed the presence of uPA, but also a pronounced heterogenecity in the content of uPA in different parts of the individual tumours. A high uPA content was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were devoid of detectable uPA. The uPA was located in the cytoplasm of the tumour cells and extracelluarly surrounding the tumour cells.
Degradation of the surrounding normal tissue is a central feature of invasiveness of malignant tumours. The constant finding of uPA in malignant tumours and the findings indicating that uPA plays a role in tissue degradation in normal physiological events have led to the assumption that uPA plays a similar role in cancer development. The hypothesis of uPA playing a role in tissue destruction involves the assumption that plasmin, together with other proteolytic enzymes, degrades the extracellular matrix. It is noteworthy in this context that most components of the extracellular matrix can be degraded by plasmin. These include laminin, fibronectin, proteoglycans, and possibly some types of collagen, but not all. In addition, plasmin can activate latent collagenases which in turn can degrade the other types of collagen (4).
Many research groups have proposed that invasive tumour cells secrete matrix-degrading proteinases and that one of the crucial cascades is the plasminogen activation system. Regulation of the proteolysis can take place at many levels including tumour cell-host cell interactions and protease inhibitors produced by the host or by the tumour cells themselves. Expression of matrix-degrading enzymes is not tumour cell specific. The actively invading tumour cells may merely respond to different regulatory signals compared to their non-invasive counterparts (10).
The assumption that the plasminogen activation system, through a breakdown of extracellular matrix proteins, plays a role in invasiveness and destruction of normal tissue during growth of malignant tumours is supported by a variety of findings. These include a close correlation between transformation of cells with oncogenic viruses and synthesis of uPA, the finding that uPA is involved in tissue destruction in many non-malignant conditions, and the immunohistochemical localization of uPA in invading areas of tumours (see (1) for review).
Further support for this hypothesis has come from studies with anti-catalytic antibodies to uPA in model systems for invasion and metastasis. Such antibodies were found to decrease metastasis to the lung from a human uPA producing tumour, HEp-3, transplanted onto the chorioallantoic membrane of chicken embryos (11,12), penetration of amniotic membranes by B16 melanoma cells (13), basement membrane invasion by several human and murine cell lines of neoplastic origin (14), and formation of lung metastasis after intravenous injection of B16 melanoma cells in mice (15). In some of these studies (13,14), a plasmin-catalyzed activation of procollagenases appeared to be a crucial part of the effect of plasminogen activation.
Urokinase-Thype Plasminogen Activator Receptor
A specific cell surface receptor for uPA (uPAR) was first detected by a saturable binding of uPA to monocytes and monocyte-like cells (16) and has since been found on many types of cultured cancer cells (17). Human uPAR is a single polypeptide-chain, highly glycosylated protein with a molecular weight of 55-60 kD (18). It is translated from a 1.4 kb mRNA (19), encoded by a single gene located on chromosome 19.
It consists of three homologous domains. The amino-terminal domain (domain 1) contains the ligand binding region (20), which binds to the EGF-like domain in the uPA molecule (21). uPAR is carboxy-terminally anchored to the cell surface by a glycosyl-phosphatidylinositol moiety (22). A possible function of this lipid anchor is to facilitate movement of uPAR on the cell membrane. uPAR can be cleaved by uPA and plasmin, releasing domain 1 and leaving domains 2 and 3 on the cell surface (23).
uPAR binds both active uPA and pro-uPA with a high affinity (K.sub.d 10.sup.-9 -10.sup.-11 M), that depends on the cell type. Pro-uPA can be activated when it is receptor-bound, and receptor-bound uPA is catalytically active (17,24). Binding of pro-uPA to uPAR and concomitant cell surface binding of plasminogen strongly enhances plasmin generation (24,25) and the surface of uPAR expressing cells are preferential sites for plasminogen activation under physiological conditions (26).
The activation of receptor-bound pro-uPA is efficiently catalyzed by surface bound plasmin leading to a strong amplification of the overall plasminogen activation reaction (24). Several other proteolytic enzymes, including plasma kallikrein (27) and cathepsin B (28) can activate pro-uPA. The physiological relevance of these latter enzymes in pro-uPA activation remains to be determined and it is still not known how the uPA pathway of plasminogen activation is initiated in vivo.
uPAR synthesis is regulated by cytokines such as TGF-.beta.1, TGF-.beta.2, EGF and by the tumour promoter phorbol myristate acetate. This regulation has in some cases been traced back to the transcriptional level, but also changes in the stability of uPAR mRNA play a role ((29,30) and L. Lund, personal communication).
Type 1 and 2 Plasminogen Activator Inhibitor
uPA activity is controlled by two specific plasminogen activator inhibitors, PAI-1 and PAI-2 (for a review see (31)). These molecules are products of different genes, located on chromosome 7 and 18, respectively. They are both glycoproteins with a molecular weight of approximately 50 kD and both belong to the serine protease inhibitor (serpin) family. PAI-1 and PAI-2 differ in their relative ability to react with uPA and tPA and also in their immunological reactivity. PAI-1 autoinactivates into a latent form but is protected from this inactivation by binding to vitronectin (32). uPA and tPA are also inhibited by protease-nexin 1, which in contrast to PAI-1 and PAI-2, also inhibits other trypsin-like proteases such as plasmin and thrombin (33). The inhibitors bind to the catalytic B-chain of active uPA. They do not react with pro-uPA. PAI-1 inhibits receptor-bound uPA nearly as efficiently as uPA in solution (24). Several cell types internalize and degrade complexes between uPA and PAI-1 or PAI-2 (34). In some cases this internalization appears to be dependent on binding to the uPA receptor (35) and recent reports indicate that the .alpha.-2-macroglobulin receptor in some cell types also plays a role in internalization of uPA/PAI-1 complexes (36). Expression of both PAI-1 and PAI-2 is regulated by a variety of cytokines, growth factors and hormones (31). The regulation of the various components of the uPA system appears to be independent of each other (9,29-31).
Localization of the Urokinase Plasminogen Activation System in Cancer Tissue
Studies of the occurrence and localization of the various components of the uPA system have shown that both uPA and uPAR are expressed at invasive foci in most experimental and human cancers that have so far been investigated. These studies have been performed both at the protein level by immunohistochemistry, and at the mRNA level by in situ hybridization. Because of the strong amplification of the proteolytic activity that is characteristic for the plasminogen activation cascade, uPA and uPAR are of very low abundancy in tissues.
In the highly invasive murine Lewis lung carcinoma, uPA protein (37) and mRNA (70) are consistently expressed by the cancer cells at invasive foci, and using a recently isolated cDNA for mouse uPAR (38) it has been found that also uPAR mRNA in this experimental carcinoma is expressed by the invading cancer cells (J.Eriksen, personal communication). PAI-1 protein is not found in the invasive areas of the Lewis lung carcinoma but is expressed by the cancer cells in non-invasive areas, suggesting that this inhibitor plays a role in protecting the tumour tissue against the proteolytic degradation (39).
An interesting finding is that Lewis lung carcinoma cells when migrating into the surrounding normal tissue without degrading it often express both uPA mRNA and PAI-1 mRNA (P. Kristensen, personal communication), suggesting that uPA activity regulated by PAI-1 is involved in migration of cancer cells. A role of uPA and PAI-1 in cell migration is also supported by a distinct localization of uPA found at cell-cell and focal cell substratum contacts in some cultured cell types. These cells also produce PAI-1 which may serve to regulate uPA activity, enabling it to act in a directional breaking up of such contacts during cell movement (72). An involvement of uPA and PAI-1 in cancer cell migration and metastasis is furthermore supported by the finding that out of 5 melanoma cell lines those 2 which produced both uPA, uPAR and PAI-1 were the most efficient in an in vitro matrix-degradation assay and most frequently produced lung metastasis in nude mice in vivo (71).
Of human cancers, colon adenocarcinomas have been most intensively studied for expression of components of the plasminogen activation system. Both uPA and uPAR are consistently present at invasive foci, but uPA protein (40) and mRNA (41) are not found in the cancer cells but in fibroblast-like stromal cells located adjacent to the invading cancer cells. uPAR mRNA (41) and protein (C. Pyke, unpublished results) are located in cancer cells and in tumour infiltrating macrophages. Therefore, in this type of cancer the malignant cells and the macrophages can presumably bind and utilize uPA released from the fibroblast-like cells. PAI-1 mRNA is in colon adenocarcinomas expressed by endothelial cells in the tumour stroma (42), while there is no PAI-1 expression in the surrounding normal tissue, suggesting that PAI-1 also in this type of cancer plays a role in protecting the tumour tissue against degradation. Another possible role of PAI-1 is to participate in the process of tumour angiogenesis (77).
In human squamous skin cancer both uPA and uPAR mRNA are expressed by the invading cancer cells (43,44). In ductal mammary carcinomas uPAR immunoreactivity is located in macrophages infiltrating the invasive foci (45), while uPA mRNA is found in adjacent fibroblast-like cells, and in some cases also in the cancer cells (43,46). PAI-1 has been detected by immunohistochemistry in endothelial cells, cancer cells and some non-malignant epithelial cells in breast cancer (47).
These studies thus show that there is a consistent expression of uPA and uPAR at invasive foci and of PAI-1 in non-invasive areas of malignant tumours as well as in endothelial cells lining the tumour vessels, and that some of the components of the uPA system are expressed by the stromal cells during cancer invasion.
A similar stromal cell expression has recently been found for several metalloproteases believed to be involved in cancer invasion (48-52) and a picture is now emerging of the stromal cells often being actively involved in the invasive process (for a recent review see (53)).
Prognostic Significance of uPA and PAI-1 in Breast Cancer
The first study which related uPA content in breast cancer tissue to patient prognosis (54) measured uPA levels by assaying the enzyme activity present in the tumour extracts and demonstrated that high activity was significantly associated with shorter disease-free interval. Subsequent studies measuring the uPA content by ELISA showed that high levels of uPA immunoreactivity was not only associated with shorter relapse-free survival but also strongly associated with short overall survival (55-58).
Janicke et al., (56) thus found in a study of 115 patients with a medium observation time of 12.5 months, that patients with high uPA levels had significantly shorter disease-free survival, the relative risk being 21.1 (95% confidence interval 2.6-174.6). This association was significant in both node-negative and node-positive patients. Similarly, Duffy et al (55) found a significant correlation between survival and uPA-level in 166 breast cancer patients with a medium observation time of 34 months, the relative risk being 11.3 (95% confidence interval 1.22-99.0).
These two studies were performed with detergent extracts of mammary cancer tissue. These extracts contain more uPA than those performed with detergent-free buffers, such as those used for routine preparation of cytosols for steroid hormone receptors (59). By the use of a combination of one polyclonal antibody preparation and three monoclonal antibodies an ELISA was constructed that readily detects uPA immunoreactivity in cytosols. Although the cytosols only contain about 12% of the optimally extractable uPA immunoreactivity, there is a close correlation between the uPA in the cytosols and the maximally extractable amount (59). With this uPA ELISA a retrospective study was performed on stored cytosols from 190 pre- and postmenopausal high risk patients who were protocolled by the Danish Breast Cancer Cooperative Group and had a medium observation time of 8.5 years (58). High cytosolic uPA was in this study significantly associated with short overall survival in both pre- and postmenopausal patients, the relative risk being 2.0 (95% confidence intervals 1.1-3.7) in the premenopausal women.
In a recent study by Foekens et al (60) uPA was assessed in breast cancer cytosol from 671 women. uPA was found significantly associated with relapse-free survival and death both in the group of node negative patients and in the group of node positive patients.
Also high PAI-1 level in mammary cancer tissue, as determined by ELISA, appears to be associated with poor prognosis (57,58). Janicke et al. (57) thus found in a study of 113 patients with a medium observation time of 25 months that patients with high PAI-1-level in their primary tumour had a significantly shorter relapse-free survival than patients with low PAI-1-level, the relative risk being 2.8 (95% confidence interval 0.98-8.3). In the Danish study of 190 high risk breast cancer patients (58), high PAI-1 level in cytosolic extracts was significantly correlated to short overall survival and short relapse-free survival in both pre- and postmenopausal patients, the relative risk with respect to overall survival being 2.9 (95% confidence interval 1.5-5.8) in postmenopausal women.
There is a positive correlation between uPA and PAI-1 levels in breast cancer tissue (47,57,58) while the two parameters in the various studies are not, or only weakly associated with, estrogen and progesterone receptor level. In multivariate analyses including established prognostic parameters, such as number of tumour positive lymph nodes, tumour size, and estrogen and progesterone receptor levels, levels of either uPA or PAI-1 are found to be independent and statistically significant prognostic parameters in most patient groups studied (55,57,58). Determination of uPA- and PAI-1 levels thus appears to add significant prognostic information to that obtained by the established parameters. It should be noted that this information, at least regarding PAI-1 levels, can be obtained from cytosols routinely prepared for steroid hormone receptor analysis.